Due to recent advances in genetic and cell engineering technologies, proteins known to exhibit various pharmacological actions in vivo are capable of production in large amounts for pharmaceutical applications. Examples of such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF), kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth differentiation factor (MGDF), osteoprotegerin (OPG), interferon (IFN), consensus interferon (CIFN), novel erythropoiesis stimulating protein (NESP), glial cell line derived neurotrophic factor (GDNF) soluble extracellular domain of tumor necrosis factor receptor fused to the Fc domain of an antibody (etanercept), antibodies to specific to various different antigens, and obesity protein (OB protein). OB protein may also be referred to herein as leptin.
The availability of such recombinant proteins has engendered advances in protein formulation and chemical modification. One goal of chemical modification is protein protection. Chemical attachment may effectively block a proteolytic enzyme from physical contact with the protein backbone itself, and thus prevent degradation. Additional advantages include, under certain circumstances, increasing the stability and circulation time of the therapeutic protein, thereby increasing. its therapeutic efficacy by reducing the ability of the immune system to detect and eliminate the therapeutic moiety.
There are several methods of chemical modification of useful therapeutic proteins which have been reported. For example, chemical modification using water soluble polymers (including, but not limited to, polyethylene glycols, copolymers of ethylene glycol/propylene glycol, polyvinyl alcohol, carboxymethylcellulose, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran) has been extensively studied, and several polymer-protein conjugate formulations having improved pharmacological properties, e.g., enhanced serum half-life, improved stability and solubility, and decreased immunogenicity have been reported.
U.S. Pat. No. 5,824,784, discloses N-terminally monopegylated granulocyte colony stimulating factor (“G-CSF”) and N-terminally monopegylated consensus interferon (“N-terminally monopegylated” denoting that the protein moiety has attached to it a single polyethylene glycol moiety at the N-terminus) which demonstrate, inter alia, increased serum half-life and improved stability.
Chemical modification with a single 20 kDa polyethylene glycol (PEG) polymer at the N-terminus of leptin results in a highly efficacious molecule which demonstrates substantial dose reduction and increased solubility relative to the unmodified native protein; see, e.g., PCT WO 96/40912.
Unfortunately, there are still a few limitations associated with certain such chemical modifications. For example, the use of polymers in chronic applications and/or in relatively large amounts, the potentially undesirable effects of the accumulation of high molecular weight, synthetic, non-biodegradable polymers are of concern. In addition, PEG-protein conjugates have been found to accumulate in kidney vacuoles when administered regularly over a period of time at high doses; see e.g., Conover et al., Artificial Organs, 21(5):369–378 (1997); Bendele et al., Toxicological Sciences, 42:152 (1997). Although it is not known if such vacuoles are detrimental to the health of an individual, it is preferable that drug administration have no associated abnormalities.
There would thus be a clear advantage for a biodegradable, biocompatible, water soluble polymeric carrier of proteins wherein the polymeric carrier can be metabolized or hydrolyzed and eventually eliminated from the body. The present invention addresses this issue and provides methods for preparing protein conjugates with a water-soluble biodegradable, biocompatible polyacetal polymer. Importantly, the polyacetal-protein conjugates described herein were biologically active and did not produce any undesirable side effects (e.g., kidney vacuole formation) in experimental animals.